Difference between revisions of "Part:BBa K3140010"

Revision as of 10:17, 21 October 2019

VVD36-C73A-CH4-M130I

VVD36-C73A-CH4-M103I is a fluoroprotein derived from the VIVID (VVD) blue-light photoreceptor in Neurospora crassa. A single cysteine to isoleucine residue substitution in VVD36-C73A-CH4 led to this part.

Usage and Biology

VIVID (VVD) is a blue-light sensing photoreceptor from the ascomycete (spore-shooting fungus) N. crassa. It is a member of a family of proteins containing a light-oxygen-voltage-sensing (LOV) domain, which modulate circadian responses to environmental stimuli^[1]. Mutation of the highly-conserved LOV domain cystine residue (Cys73) to alanine will convert VVD into a fluoroprotein. In addition, previous work ^[2] indicates that truncation of the first 36 amino acids of VVD increases its stability in heterologous systems. Our VVD part incorporates both of these changes.

VVD36-C73A-CH4-M103I is a 456 bp sequence, encoding a 151 amino acid peptide (17.2 kDa). We cloned VVD36-C73A, VVD36-C73A-CH1, VVD36-C73A-CH2, VVD36-C73A-CH3, and VVD36-C73A-CH4 into pK18, which were then transformed into Escherichia coli TOP10 cells. In order to validate the insertion of these genes, we conducted a PCR with primers that were specific to the pK18 backbone, which should a 624 bp product. This was verified with agarose gel electrophoresis, demonstrating had the expected size (Fig. 1).

Fig. 1: Agarose gel electrophoresis of VVD36-C73A, VVD36-C73A-CH1, VVD36-C73A-CH2, VVD36-C73A-CH3, VVD36-C73A-CH4, and VVD36-C73A-CH4-M130I conducted on 1% agarose gel in 1X TAE for 50 min at 100 V.

We conducted a fluorescence assay using a TECAN Spark microplate reader Escherichia coli TOP10 cultures containing VVD36-C73A, VVD36-C73A-CH1, VVD36-C73A-CH2, VVD36-C73A-CH3, VVD36-C73A-CH4, and VVD36-C73A-CH4-M130I in pK18 (Fig. 2). As shown, VVD36-C73A-CH4-M130I exhibits the greatest fluorescence, followed by VVD36-C73A-CH2. All other VVD38-C73A variants were only slightly more fluorescent than control.

Fig. 2: Graph of TECAN OD-normalised fluorescence for Escherichia coli TOP10 cultures containing VVD36-C73A, VVD36-C73A-CH1, VVD36-C73A-CH2, VVD36-C73A-CH3, VVD36-C73A-CH4, and VVD36-C73A-CH4-M130I in pK18.

When these cultures were plated, it was immediately apparently that VVD36-C73A-CH4-M130I had strong fluorescence compared to the other VVD36-C73A variants (Fig. 3).

Fig. 3: VVD36-C73A-CH4-M130I compared to VVD36-C73A, VVD36-C73A-CH1, VVD36-C73A-CH2, VVD36-C73A-CH3, and VVD36-C73A-CH4 under long-wave UV.

Sequence and Features

1. ↑ Schwerdtfeger, C. & Linden, H. VIVID is a flavoprotein and serves as a fungal blue light photoreceptor for photoadaptation. EMBO J 22, 4846-55 (2003).
2. ↑ Zoltowski, B.D. et al. Conformational switching in the fungal light sensor Vivid. Science 316, 1054-7 (2007).